This invention relates to vaccines including oral vaccines against chlamydial antigens, and a process for making the vaccine.
Chlamydial infection is a diverse group of conjunctival, genital, respiratory, and neonatal infections occurring primarily on mucosal surfaces. The etiologic agent of the infection is an obligate intracellular bacterial parasite of eukaryotic cells, chlamydiae. There are three genetically different species in this genus, with certain similarities in morphology, intracellular developmental cycle and antigenic responses: Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae.
The infection by C. trachomatis is limited to humans. Fifteen serovars are differentiated based on the antigenic variations of the major outer membrane protein (MOMP) (Grayston and Wang, J. Infect. Dis., 132:87, 1975). Serotypes D-K, are the most common cause of sexually transmitted venereal diseases. Conservatively, more than 4 million cases of chlamydial sexual infections occur each year in the United States making it more prevalent than all other sexually transmitted diseases combined. The diseases include nongonococcal urethritis, mucopurulent cervicitis, acute epididymitis, ectopic pregnancy and pelvic inflammatory disease (PID, endometritis, salpingitis, parametritis and/or peritonitis). The infection in women can be quite damaging: Among 250,000 cases of pelvic inflammation diseases caused by this organism in the U.S. each year, 10% lead to infertility. When infants are born to chlamydia-infected mothers, they are at high risk of developing inclusion conjunctivitis and pneumonia. C. trachomatis serovars A, B, Ba, and C cause trachoma, an infection of conjunctival epithelial cells. The chronic and secondary infections induce the infiltration of subepithelial lymphocytes, forming follicles and the invasion of fibroblasts and blood vessels to the cornea, leading to blindness. On the other hand, the formation of the scar and malformation of the eyelid, causing trichiasis' constant scraping of the cornea by the eyelash can also lead to corneal opacification and blindness. There are approximately 500 million trachoma cases in the world, and between 7 and 9 million are now blind because of its complications making it the world's leading cause of preventable blindness. The prevalence of active trachoma is high in early age. There are 80 million children in need of treatment. It has been an enormously important health problem in the Middle East, North Africa, South Asia and North India.
C. psittaci mainly affects animals and birds. It had, and still has a great economic impact in dairy, wool and meat industries. There are 9 serovars from mammalian species, 7 serovars from avian species and 2 biovars from koala bears. Mammalian serovar 1, 2, 3, and 9 infect cattle and sheep, causing a wide range of disorders from placenta and fetus infection and other reproductive problems, including polyarthritis-polysisitis, encephalomyelitis, conjunctivitis as well as intestinal infections. Although numerous attempts have been made to produce vaccines, only modest success has been achieved (Schnorr, J. Am. Vet. Med. Assoc. 195:1548, 1989). Serovars 4, 5, and 6 are the causes of abortions, pneumonia and polyarthritis in porcine species. Serovar 7 represents chlamydial strains of feline conjunctivitis, rhinitis and pneumonitis and serovar 8 includes guinea pig inclusion conjunctivitis. The avian strains often cause human infection in bird handlers and poultry processing workers.
C. pneumoniae is a newly identified species. To date, one serovar has been identified, TWAR (Grayston, Proceedings of the Seventh International Symposium on Human Chlamydial Infections, Pg. 89, 1990). Current evidence suggests that C. pneumoniae is a primary human pathogen that is transmitted from human to human and causes about 10-20% of community acquired pneumonia in adults. It has become the main causative agent of human respiratory diseases such as pneumonia, bronchitis, pharyngitis, and sinusitis and a possible agent in reactive arthritis. Epidemics have occurred in hospitals, in the military and families. The serological finding from many countries have shown that 50-55% of adults with antibody against TWAR antigen are specific for C. pnueunoniae. It is the major bacterial cause of illness in newborn. The infection to elderly persons and those with chronic diseases may cause serious illness or even death.
The pathogenicity of chlamydial infection is not well understood. It is long known that different individuals infected by these serovars exhibit different clinical manifestations. It has been proposed that it was likely due to the variation of the host immune response. It has been shown that immunologic response to the synthetic Th/B cell epitopes in the various inbred strains of mice is different, indicating that the T helper epitope is recognized in the context of the multiple major histocompatibility complex.
The target of chlamydial invasion are typically epithelial cells of a host. It is still not certain how the chlamydial elementary bodies (EB), (a sporelike, spherical particle, about 300 nm in diameter), enter the host cell: receptor-mediated endocytosis, and/or non-specific high affinity absorption. It has been reported that two proteins, 18 and 32 kD of C. trachomatis bind to Hela 299 cell membrane preparations. Recently, another heat-labile protein membrane protein, 38 kD, was proposed a binding to Hela cell line, suggesting a ligand like mechanism. It has also been proposed that since chlamydia have the ability to infect a wide variety of mammalian cells in vitro, there must be some adherence mechanism for the establishment of the infection. The major outer membrane protein was proposed as such an adhesin. Recently, it has been demonstrated that a heparin sulfate-like glycosaminoglycans present on the surface of chlamydia organisms is required for attachment to host cells. The receptors on the host cells have also been studied. It was suggested that proteins, 18,000 and 31,000 kD from Hela cells are the receptors due to trypsin sensitivity for the EB specific binding. It also has been shown that C. trachomatis and C. pneumoniae bind specifically to a lipid on Hela cells. Nuclear magnetic resonance spectroscopy analysis and atom bombardment mass spectrometry show that it was phosphatidylethanolamine (PE). At the same time ganglia-series glycolipids were found specifically bound to EBs. All those findings suggest that the mechanism of endocytosis by epithelial host cells is still a matter of uncertainty. Once the EBs enter the host cells by endocytosis, depending upon conditions, they are transformed into a metabolically active, non-infectious reticulate body (RB). The prime purpose of RBs is intracellular replication by binary fission using host metabolites. This occurs in a membrane-bounded vesicle, termed an inclusion. This inclusion (endosome) can resist the fusion with the lysosomes of host cells. Each RB eventually gives rise to one or more EBs which can initiate another infectious cycle. Host cells may be lysed by release of inclusion bodies or undamaged by exclusion body exocytons. Surface antigens are thought to direct both phagocytosis and evasion of phagolysosomal fusion.
The treatment of chlamydial infections has relied on the administration of antibiotics. This has been proved effective in the early stages of the infection depending upon proper timing for diagnosis and screening. The problem is that the infection can be asymptomatic. Most patients don't realize its presence until it has occurred for a period of time. In the chronic stage as in the case of genital infection, it has been demonstrated that little can be done to prevent the damage of the reproductive tracts in a monkey infection model.
Vaccines employing the whole organism or sub-units of the organism have been used in an attempt to prevent chlamydia infections caused by members of the trachoma biovars. These attempts, however, have been disappointing, partially due to host hypersensitivity in reaction to the vaccines (Grayston et al, Clini. Med. J. (Republic of China) 8: 312, 1961, Wang et al, 1967, Am. J. Opthalmol. 63: 1615, Schachter, Pathol. Immunopathol. Res. 8: 206, 1989). The pathogenesis associated with infections believed to be a process of delayed hypersensitivity. It is thought that chronic inflammation resulting from repeated reinfection of humans have an important role in the conjunctival infiltration, blinding sequelae of trachoma and scarring of the fallopian tubes which result in infertility and ectopic pregnancy. The surface antigens of elementary bodies have been the focus of research attempting to identify a protective antigen.
Surface components of chlamydia actively interact with host cells and with the host's immune system. They are believed to account for the attachment, endocytosis and the immune response, but the exact nature and regulation of these interactions has not yet been fully identified. Several distinct antigenic components of C. trachomatis, C. psittaci and C. pneumonia have been investigated including the identification, characterization and function in chlamydial infection. Moreover, it is of importance to determine the mechanism of infection and determine the protective antigens. Surface exposed antigens are the main targets of much research since they are accessible to the immune or other defense systems. The antigens most actively investigated include major outer membrane proteins (MOMP), chlamydial lipopolysaccharide, 60-kD heat shock protein (HSPO 60) adhesions and a glycolipid exoantigen termed the exoantigen (GLXA).
In the outer membrane of chlamydia there are three cysteine-rich proteins 57, 40, and 12.5 kD which resembles the matrix proteins of gram-negative bacteria. The 57 and 12.5 kD proteins can not be found in the replicating form of the bacteria RBs. As the major outer membrane protein (MOMP), 40 kD, is abundant in both infectious EBs and RBs. In RBs, the protein could function as pore-forming proteins that permit exchange of nutrients for the reticulate bodies. Genetic and molecular characterization have shown that this protein is composed of four variable segments (VS) interspersed among five constant segments. Those variable segments are surface exposed and have the determinants of serovar, subspecies and species specificity.
The studies on immune responses to this protein are mostly carried out by immunization of animals with purified protein. In vitro neutralization experiments have been conducted using the mixture of poly or monoclonal antibodies specific to MOMP and EBs to infect cell culture. These experiments indicate that the antibodies specific to MOMP protein or one single epitope prevent the inclusion bodies formation in cell culture. The mechanism of the neutralization does not involve inhibition of the attachment or penetration, but rather interfere with the process after internalization. Using monoclonal antibodies generated by the whole elementary bodies of serovar B, the monoclonal antibodies which recognized the immuno-accessible MOMP epitope in dot blot assays, neutralized the infectivity of organisms of monkey eyes. The protection was serovar-specific. In a later experiment by using Fab fragments of the monoclonal antibody, it has been further demonstrated that monovalent Fab neutralized the infection by preventing the attachment to Syrian hamster kidney (HaK) cells. Confirming that the protection is not due to the aggregation of bivalent IgGs. T cell epitopes of MOMP have also been investigated. T cell proliferation responses were found in splenic T cells obtained from A/J mice immunized with MOMP in the presence of overlapping synthetic peptides which represent primary sequence of serovar A MOMP. The synthetic peptides which produced T cell response correspond to surface-accessible serovar-specific epitopes located in variable domains (VD)VD I and VD IV. By using a similar approach, it was found in BALB/cByJ mice that VDIII fragment is T cell dependent. It also has been shown that by using chimeric T/B cell peptides derived from two epitopes of MOMP, one is a conserved T helper cell epitope and the other is a serovar A specific neutralizing epitope. Some mice immunized with this peptide produced high-titered serum-neutralizing antibodies, while others did not. Although MOMP has been a most intensively studied surface antigen and the neutralization antisera has been produced in experimental animals, there are still many unsolved questions regarding the immune response. For example, the neutralization of infection is serovar specific, thus, it is limited as a vaccine candidate. The neutralization is still limited to in vitro studies, and there has been no convincing in vivo protection from challenge by immunization with any MOMP or chimeric epitopes known.
It has long been known that a chlamydia genus specific antigen was a glycolipid (Dhir et al, J. Immunol. 109: 116-122, 1972). Much later, it was found that lipopolysaccharide (LPS) was in the outer membrane of both EBs and RBs of chlamydia. It has a chemical structure similar to enterobacterial LPS of the Re chemotype (Nurminen et al, Science, 220: 1279-1281, 1983). The monoclonal antibodies prepared by immunization with EBs of serovar L2 were specifically against LPS of chlamydia, but not LPS from N. Gonorrhoeae, S. typhimurium, or E. coil. However, antibodies produced by S. typhimurium Re LPS or lipid A recognized chlamydial LPS (Caldwell and Hitchcock, Infect. Immun., 44: 306, 1984). This demonstrated that chlamydial LPS has an unique antigenic domain compared to other gram-negative bacteria. Further characterization has shown that a chlamydia-specific domain contained in its saccharide portion, 3-deoxy-D-manno-2-octulosonic acid (KDO) with a sequence of KDo (2-8)-KDo (2-4)--KDo(KDo.sub.3) The 2.8 linked moiety is the structure characteristic of chlamydia LPS. Studies have also been carried out in the distribution and the relocation of LPS on the outer membrane during the developmental cycle. By immunostaining with a monoclonal antibody it was shown that LPS is loosely bound in the membrane during the developmental cycle, and not shed into media.
Chlamydial LPS was thought to be an ideal antigen for the vaccine candidate because of its abundance on the surface and its being antigenic. LPS was suspected as an important virulent determinant in the early steps of the infection and the antibodies specific to it serve some function in resolving the chlamydial infection. However, little is known concering the biological function of LPS or the immunological respone to it. It appears that the antibodies which are specific to LPS only have been useful in the diagnosis of chlamydial infection and location of LPS, but not effective in resolving an infection.
Other genus-specific chlamydial antigens are 57 to 60 and 75 kD proteins which have been identified as related to the heat shock protein (HSP) family. This was done by comparing the sequence of the operons encoding these proteins to the groE stress response operon of E. coli or B. megaterium. The antigenic identity of 57 kD protein was confirmed by the reaction with anti-HSP-60 antiserum. The 60 kD protein elicited an ocular hypersensitivity response in immune guinea pigs, which was characterized by a predominantly mononuclear macrophage and lymphocyte cellular infiltration (Watkins, et al, Proc. Natl. Acad. Sci. U.S.A. 83: 7580, 1986, Morrison et al, S. Exp. Med. 169: 663, 1989). This was the first indication that an antigen is responsible for delayed hypersensitivity in chlamydial ocular infection. The precise involvement of this protein in stimulating immunopathogenic responses in human chlamydial diseases has not been determined. There is evidence that shows a certain percentage of sera taken from women with PID, ectopic pregnancy and tubal infertility have high anti-chlamydial antibodies reacting to chlamydial HSPO-60 heat-shock protein. However, not every patient serum which has high titer to chlamydia reacts with it, indicating that either HSP-60 is not surface exposed or antigenicity is MHC restricted.
The 75-kD protein was found preferentially transcribed during heat stress of chlamydial organisms. The monospecific antibodies from rabbits raised against 75-kD protein were found to bind to the organism and neutralized the infection in vitro. It is an exposed antigen in the outer membrane.
Genus-specific glycolipid exoantigen (GLXA) was originally isolated from the supernatants of chlamydia infected cell cultures (Stuart and MacDonald, Current Micobiology, 11: 123, 1982). It has been characterized chemically, biologically and serologically in recent years. (Stuart and MacDonald, Proceedings of the Sixth International Symposium on Human Chlamydia Infections, p167, 1986, Stuart et al, Immunology, 67: 527, 1987). Mass spectrographic analysis indicated that GLXA contains polysaccharides: gulose, (not glucose), mannose and possibly galactose, while the lipid component has fatty acids of chain length C17 and C18:1. There is no KDO or lipid A found in its structure. It is produced and released from the infected cells during the growth cycle in vitro. Transmission electron microscopy utilizing colloidal gold-conjugated goat anti-mouse second antibody to detect the specifically bound monoclonal antibody revealed that GLXA is mostly extracellular 48 hours after the infection (Stuart et al, Immunolgy, 74: 747, 1991) which is different from that found for chlamydial LPS. Human sera from patients with clinically defined lymphogranuloma venereum (LGV) contain IgG antibodies which recognize GLXA (Stuart and MacDonald, Immunology, 68: 469, 1989), demonstrating the immunoaccessibility in the natural infection. But, there was little information on its function in the chlamydial infection and the immune response to it. The overall immunological reaction to chlamydial antigens is not well understood. It is still not known how the chlamydia evade the host immune surveillance. Antibodies found specific to chlamydial antigens in infected human patients have shown little protection for later infection. Although chlamydia mainly affects mucosal surfaces, the clinical relevance of the IgA immunity to it has not been completely described. The feasibility of chlamydia vaccine depends on producing a protective host defense which may include S-IgGA response, a cell mediated response and possibly a humoral antibody. In addition, the ability to produce large quantities of this antigen indicates a synthetic and/or chimeric antigen may be the method of choice.
Idiotypes have been intensively studied following Jerne's network theory in 1974. One of his major proposals is the self-regulation of the immune system through a network of idiotype-anti-idiotype interactions (Jerne, 1974). It is suggested that the idiotopes on a single antibody molecule can mimic (that is, be the "internal image") of any foreign or self epitope at the molecular level.
All idiotypes of a single immunoglobulin molecule have been found to be located on Fv (fragment variable) region by studies showing that the inhibition of binding of anti-idotypic antibodies to the idiotype is the same between Fv and Fab (Givol, 1991). In general, anti-idotypic antibodies are divided into three types Ab.sub.2 .alpha., Ab.sub.2 .beta. and Ab.sub.2 .epsilon.: Only Ab.sub.2 .beta. binds to the complementary determining region, thus can be the internal image of the antigen. The occurrence of Ab.sub.2 displaying internal image of properties must adhere to the following criteria; (1) binding onto Ab.sub.1 and to any other anti-nominal antigen antibodies from another species and lack of reactivity with Ab.sub.2 to other antibodies; (2) inhibition of the binding of Ab.sub.1 to the specific antigen, the nominal antigen, and (3) the ability to elicit the synthesis of Ab.sub.3 with anti-antigen specificity in animals without previous exposure to the antigen (Ertl and Bona, 1988).
The important role of anti-idiotypic antibodies in vivo has been shown in numerous experiments. The administration of anti-idiotypic antibodies was found to elicit different effects: either suppression or enhancement of the responses to the specific idiotype (Hart, 1972, Kennedy, 1983). In autoimmunity, it certainly plays an important role. The pathology associated with many autoimmune diseases is most likely due to (at least in part), a direct idiotype-anti-idiotype interaction of the autoimmune antibodies with anti-idiotypic antibodies. Idiotypic specificity in a specific antibody were first characterized, by demonstrating that specific hapten binding could inhibit idiotype recognition. The first experimental support for the validity of the internal image was presented by Sege and Peterson in 1978 by using anti-idiotype as a probe to identify cell surface receptors.
The best information for the exact molecular basis for the mimicking presently is obtained from the X-ray crystallography of the idiotype-anti-idiotype complex. The basis of molecular mimicry of the antibodies can be either local sequence homology to the original protein as in a reovirus system or, in most cases, identical conformations from entirely different amino acid sequences as in the hemoglobin-myoglobin family of proteins. X-ray crystallography and sequence data in the later studies showed that identical, functional conformations can be assumed by proteins that differ by as many as 137 of 141 amino acids. The studies of the crystal structure of idiotope-anti-idiotope complex in the anti-lysozyme antibody and the anti-idiotope have demonstrated that a private idiotope consists of 13 amino-acid residues, most from the complementarily-determining regions, but including three residues from the third framework region of its VL domain. Seven of these residues are common with the paratope of anti-lysozyme antibody, indicating a significant overlap between idiotope and antigen-combining site. Idiotype has been a unique tool in characterization and manipulation of the immune response since it was found and realized: as a clonal marker to follow B cell development, somatic mutation and fate of clones of B cells. They have been used as a phenotypic marker for germ-line V genes. Anti-idiotypic antibodies which bear the internal image of external pathogens such as virus, bacteria or parasites have been used as surrogate antigens for vaccine and are being used in treating B cell lymphoma and autoimmune disease such as encephalomyelitis. In addition, it has been shown that anti-idiotypic antibodies can induce T-cell responses in which either by toxic T-cells or T-helper cells are produced which recognizes the original antigen.
The provision of a protective vaccine against chlamydial infections of the eye, genital tract, lung or heart would have worldwide public health benefit. The requirements for a successful vaccine candidate against C. trachornatis must include: (a) immunogenicity after presentation in an clinically safe carrier, (b) induction of neutralizing anti-chlamydial antibody, (c) reduction of clinical and histopathologic disease, and (d) absence of chlamydia-specific delayed hypersensitivity. Several chlamydial antigens have been shown to be immunogenic in patients and animal models. The major outer membrane protein (MOMP) has been the favored candidate antigen because of its immunogenicity, and the demonstration of neutralizing anti-MOMP monoclonal antibodies. However, various formulations of MOMP ranging from membrane extracts to fusion proteins containing MOMP subunits have been variably immunogenic, but none have protected against disease in the monkey model of trachoma.
Prior to the present invention, production of neutralizing antibodies by using anti-idiotypic idiotype antibody to mimic carbohydrate antigen have been produced in a bacteria system, Schriver et al, J. of Immunol; 144: 1023, 1990.
Accordingly, it would be desirable to provide a means for preparing a genus specific oral vaccine capable of providing immunization from chlamydial infection. It would also be desirable to provide a means for producing such an oral vaccine in quantity and to provide a process for increasing the effectiveness of the vaccine.